Neuroprotective agents

ABSTRACT

Compounds of formula (I) wherein: Q represents an amidino group, a cyano group or a group of formula XYN—, (where X and Y are hydrogen or various groups); R a  represents alkylene; R b  and R c  each represents alkylene, the total number of carbon atoms in said straight chains of R b  and R c  being 7); R 2  and R 3  each represents hydrogen, or a group of formula R, RCO—, ROCO—, or RNHCO—, where R represents alkyl or aryl; the chiral carbon atom indicated by the asterisk is in the L configuration; Z is an aromatic amino acid residue; n is 0 or 1; R 1  represents hydrogen, alkyl or aryl; and W represents hydrogen, alkyl or aryl; and pharmaceutically acceptable salts thereof have the ability to protect against the neuronal damage which may be caused by an ischemic event.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 09/581,397,filed Oct. 2, 2000, now U.S. Pat. No. 6,797,699, which is a 371 ofPCT/GB98/03775, filed Dec. 16, 1998, which claims priority to U.K.application 9726569.8, filed Dec. 16, 1997.

The present invention relates to neuroprotective agents.

In events such as prolonged hypoxia and ischaemia, which may or may notbe associated with hypoglycaemia, neuronal damage, to varying degrees,is encountered.

Ischaemia typically occurs during heart attacks, but the damage incurredat these times is substantially limited to the heart tissues, andcertain treatments have been developed. With regard to the presentinvention, we are concerned with the effects of more long term ischaemiaon the brain, such as occurs with stroke patients or as a result of headinjury. The severity of the ischaemia depends on the nature of thestroke or injury, but, invariably, there is brain damage, and it is thiswhich the present invention addresses.

Various neuroprotective agents are known in the art which attempt toalleviate the problem of brain damage, but all of those currently knowntend to be associated with adverse side effects. For example, MK801(dizocilpine maleate) is a fairly simple molecule and is known toprovide a level of neuroprotection to ischaemic patients. However, MK801is also associated with “alarming psychotropic effects” (Martindale), aswell as adverse motor effects. The neuroprotective effects are detailedin Brain Research 755 (1997) 36–46 (Pringle, A. K., et al), incorporatedherein by reference. These same authors also described theneuroprotective effects of conotoxin in an earlier paper but, despitethe neuroprotective effects of this compound, adverse side effects, invivo, are observed.

Recently, research has been performed on a series of polyamine compoundsrelated to spermidine, and these compounds are disclosed in WO93/12777,with specific reference to their use as cationic channel regulatingagents. These compounds are disclosed in connection with methods forregulating cation transport across cellular membranes possessing cationchannels, the compounds being polyamine compounds having a lysine orarginine-based moiety (or a guanidine moiety) coupled to a straightchain polyamine. Mention of their effect on NMDA (N-methyl-D-aspartate)receptors is also made. These compounds were unpredictable in theireffect on cationic channels, various compounds having an effect onP-type calcium channels, whilst other compounds had effects on potassiumand sodium channels. Although these compounds have subsequently beenused in research for their effects on calcium channels, researcheffectively finished with the publication in Proc. Natl. Acad. Sci. USA[86, 1689–1693 (1989), Llinàs, R, et al], which disclosed that asubstance known as FTX from funnel-web spider toxin was toxic to mice inextremely small doses.

The present inventors were not aware of the research by Llinàs and hiscolleagues, and were pursuing similar compounds, as they were known tohave some calcium channel blocking activity. In fact, what wasdiscovered was that, not only is the calcium channel blocking activitynot very significant, but also there is little or no effect on NMDAreceptors. Further, it was also established that these compounds are,despite the earlier research, non-toxic, and they also have asubstantial neuroprotective effect.

It is believed that the reason for the discrepancy between the earlierresults and the present results lies in the preparation of thecompounds. In particular, the FTX component of funnel-web spider toxinwas specifically isolated from the toxin in the prior art, rather thanbeing prepared separately. This compound is currently thought to havethe following formula (1)

Related compounds have been manufactured synthetically, using theapproaches described herein, which result in little or no detectablecontamination of the end product. The results in the various assayshave, therefore, been exceedingly surprising in that the compounds haveproven non-toxic, as well as to have little effect on calcium channels.Indeed, if there were a substantial effect on P-type calcium channelsand/or the compounds were toxic, then there would be no use for them inthe clinical field. Instead, we find that the compounds, in theirpurified form, have use as neuroprotective agents.

Thus, in a first aspect, the present invention provides a substantiallypure compound having the general formula (I)

wherein:

-   -   Q represents an amidino group, a cyano group or a group of        formula XYN—, where        -   X and Y are the same or different, and each may represent a            hydrogen atom, a lower alkyl group, or a simple hetero-atom            containing group or, together with the nitrogen atom to            which they are attached, form a nitrogen-containing            heterocyclic group;    -   R^(a) represents a straight or branched chain alkylene or        alkenylene group having from 1 to 6 carbon atoms and each        optionally being substituted by from 1 to 4 alkyl groups each        having from 1 to 3 carbon atoms;    -   R^(b) and R^(c) each represents an alkylene or alkenylene group        having 3 or 4 carbon atoms in a straight chain, each being        optionally substituted by 1 or 2 alkyl groups each having from 1        to 3 carbon atoms, the total number of carbon atoms in said        straight chains of R^(b) and R^(c) being 7;    -   R² and R³ are the same as or different from each other and each        represents a hydrogen atom, or a group of formula R, RCO—,        ROCO—, or RNHCO—, where        -   R represents a lower alkyl group or an aryl group, said            alkyl or aryl group being optionally substituted by one or            more of the substituents α, defined below;    -   the chiral carbon atom indicated by the asterisk is in the L        configuration;    -   Z is an aromatic amino acid residue;    -   n is 0 or 1;    -   R¹ represents a hydrogen atom or a lower alkyl group or an aryl        group, said alkyl or aryl group being optionally substituted by        one or more of the substituents α, defined below; and    -   W represents a hydrogen atom or an alkyl or aryl group;        and pharmaceutically acceptable salts thereof.

A preferred class of compounds of the present invention are thosecompounds of formula (Ia):

(wherein Q, R^(a), R^(b), R^(c), R², R³, Z, n, and R¹ are as definedabove) and pharmaceutically acceptable salts thereof.

A still more preferred class of compounds of the present invention arethose compounds of formula (Ib):

wherein:

-   -   X, Y, Z, n and R¹ are as defined above;    -   x is an integer from 1 to 5;    -   y is 3 or 4    -   R⁴, R⁵, R⁶ and R⁷ may be the same or different and each        represents a hydrogen atom or a lower alkyl group; and    -   the chiral carbon atom indicated by the asterisk is in the L        configuration;        and pharmaceutically acceptable salts thereof.

Substituents α are selected from: halogen atoms, amino groups,alkylamino groups, dialkylamino groups, cyano groups, hydroxy groups,alkyl groups (except when the substituted group is alkyl), aryl groups,carbamoyl groups, alkylcarbamoyl groups, dialkylcarbamoyl groups andcarboxy groups and esters thereof.

The present invention further provides non-toxic compounds of formula(I), (Ia) or (Ib) as defined above. There is still further provided aneuroprotective composition comprising a compound as defined above, aswell as use of a compound as defined above in the manufacture of amedicament for the retardation of neuronal damage before, after orduring an ischaemic event. The invention also provides a method oftreating a mammal, which may be human, to protect said mammal from theneuronal damage caused by an ischaemic event by administering to saidmammal before, after or during an ischaemic event an effective amount ofa non-toxic compound of formula (I), (Ia) or (Ib) as defined above.

By substantially pure is meant a compound which, under conditions ofHPLC (high performance liquid chromatography) is not shown to have anyor any significant amount of contaminants detectable thereby. Tracelevels of contaminants may be acceptable in certain circumstances andsuch circumstances may be determined by the skilled person at the time.In general, levels of contaminant should be less than 1%, and preferablysubstantially less than 1%, for example less than 0.1%, possibly as lowas 0.001%.

In the alternative, it is preferred that the compounds are non-toxic, bywhich is meant that the compounds should not exhibit any unacceptablelevels of toxicity at the dosages at which they are applied. Preferably,they should exhibit no toxicity whatsoever.

Regardless of the foregoing, the class of compounds defined above isuseful for neuroprotection under hypoxic or ischaemic conditions, and wehave demonstrated this by tests on the hippocampus, as described below.The levels at which these compounds are active are substantially lowerthan those at which the prior art compounds are active.

The compounds of the present invention may be applied to the patient ifit is suspected that they are in danger of an ischaemic event,especially a stroke or head injury. Such prophylactic application may beexceedingly useful. However, it has also been demonstrated that thecompounds of the present invention have useful activity, even if appliedafter an ischaemic event, but it will be appreciated that it ispreferred to administer the compounds as soon as possible, in order toavoid as much neuronal degeneration as possible. In some circumstancesit may be desirable to administer repeated doses, especially where thepatient remains in danger of an ischaemic event.

Suitable methods of administration are generally by injection; in orderto achieve the desired result as soon as possible. Thus, intravenousinjection is particularly preferred but, in some circumstances it may bepreferable to administer the compound directly into the cerebrospinalfluid.

The dose of the compound of the present invention will vary dependingupon many factors, including the age, body weight and general conditionof the patient, as well as the mode, frequency and route ofadministration. However, a dose of from 0.01 to 50 mg/kg body weight isgenerally recommended, a dose of from 0.05 to 20 mg/kg body weight beingmore preferred. This may be administered in a single dose or in divideddoses.

In the compounds of the present invention, it is generally preferredthat the overall length of the compound is in the region of the lengthof Compound A, as shown hereafter. Compound A can be considered to be 18units long, so that we prefer the compounds of the present inventionshould be no longer than 25 units long, and no shorter than 14 unitslong. This is a general preference, but it is generally noted that thereis a rapid drop-off in activity with a length change of anysignificance, even one unit having a generally undesirable effect.Accordingly, it is more preferred that the compound should be from 17 to22 units long. By “unit” is meant an atom in the longest chain,excluding hydrogen, and those non-chain atoms attached thereto. Thus,for example, in formula (Ia), the group —NH₂ is regarded as a unit, asare the groups CR²R⁴, CO, CR⁴R⁶, etc.

Q may represent a cyano group, an amidino group or a group of formulaXYN—.

Where X or Y represents a lower alkyl group, this preferably has from 1to 6 carbon atoms and may be a straight or branched chain group havingfrom 1 to 6, preferably from 1 to 4, carbon atoms. Examples include themethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,pentyl, isopentyl, neopentyl, 2-methylbutyl, 1-ethylpropyl,4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl,3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl,1,2-dimethylbutyl, 1,3-dimethyl-butyl, 2,3-dimethylbutyl, 2-ethylbutyl,hexyl and isohexyl groups. Of these, we prefer those alkyl groups havingfrom 1 to 4 carbon atoms, preferably the methyl, ethyl, propyl,isopropyl, butyl and isobutyl groups, and most preferably the methylgroup.

Where X or Y represents a simple hetero-atom containing group, this maybe an acyclic or cyclic group. Examples of acyclic groups include theamidino group (to form, with the nitrogen atom to which X and Y areattached, a guanidino group), alkoxycarbonyl groups (to form analkoxycarbonylamino group), the carbamoyl group or thiocarbamoyl group(to form the ureido group or the thioureido group). Examples ofheterocyclic groups which may be represented by X and Y include thosegroups having from 5 to 10 ring atoms (in one or two rings), of whichfrom 1 to 4 are nitrogen and/or oxygen and/or sulphur hetero-atoms, theremainder being carbon atoms. Where there are 4 hetero-atoms, we preferthat all 4 are nitrogen atoms. Where there are 3 hetero-atoms, we preferthat all 3, 2 or 1 are nitrogen atoms. Where there are 2 hetero-atoms,we prefer that 2 or 1 are nitrogen atoms. Examples of such groupsinclude the pyrrolyl, tetrazolyl, indolyl, thiazolyl, furyl, pyranyl,chromenyl, imidazolyl, pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl,pyridyl, pyrazinyl, pyrimidinyl, isoindolyl, quinolyl, isoquinolyl,carbazolyl, chromanyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl,piperidyl, piperazinyl, indolinyl and morpholinyl groups.

Alternatively, X and Y, together with the nitrogen atom to which theyare attached, may form a nitrogen-containing heterocyclic group.Examples of such heterocyclic groups include those groups having from 5to 10 ring atoms (in one or two rings), of which from 1 to 4 arenitrogen and/or oxygen and/or sulphur hetero-atoms, the remainder beingcarbon atoms. Where there are 4 hetero-atoms, we prefer that all 4 arenitrogen atoms. Where there are 3 hetero-atoms, we prefer that all 3, 2or 1 are nitrogen atoms. Where there are 2 hetero-atoms, we prefer that2 or 1 are nitrogen atoms. Examples of such groups include the1-pyrrolyl, 1- or 2-tetrazolyl, 1-indolyl, 3-thiazolyl, 1-imidazolyl,1-pyrazolyl, 2-isothiazolyl, 3-oxazolyl, 2-isoxazolyl, 1-pyridyl,1-pyrazinyl, 1-isoindolyl, 1-quinolyl, 2-isoquinolyl, 9-carbazolyl,1-pyrrolidinyl, 1-pyrrolinyl, 1-imidazolidinyl, piperidino,1-piperazinyl, 1-indolinyl and morpholino groups.

Where Q represents an alkoxycarbonylamino group, the alkoxy partpreferably has from 1 to 6 carbon atoms and may be a straight orbranched chain group. Examples of such groups include themethoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino,isopropoxycarbonylamino, butoxycarbonylamino, pentyloxycarbonylamino andhexyloxycarbonylamino groups, of which we prefer those groups havingfrom 1 to 4 carbon atoms, and most prefer the ethoxycarbonyl aminogroup.

Preferably at least one of X and Y represents a hydrogen atom. Weparticularly prefer that one or both of X and Y represents a hydrogenatom. Particularly preferred compounds are those compounds of formula(I) in which both X and Y represent hydrogen atoms or those in which oneof X and Y represents a hydrogen atom and the other represents anamidino group or a carbamoyl group. The most preferred compounds arethose compounds of formula (I), (Ia) and (Ib) in which both X and Yrepresent hydrogen atoms or those in which one of X and Y represents ahydrogen atom and the other represents an amidino group.

The length of the groups represented by R^(a) and R^(b), that is, informula (Ia), the size of x in combination with y, is not particularlyimportant, except that the preferred overall length of the compound ispreferably observed. Whilst any particular alkylene or alkenylene grouprepresented by R^(a) may be as much as 6 carbon atoms long, it ispreferred to restrict each alkylene chain to no more than 5, butpreferably 3 or 4, carbon atoms, and an overall combination oftrimethylene and tetramethylene groups is generally preferred. Examplesof such alkylene and alkenylene groups include the methylene, ethylene,trimethylene, tetramethylene, pentamethylene, hexamethylene, vinylene,propenylene, but-1-enylene, but-2-enylene, pent-1-enylene,pent-2-enylene, pent-3-enylene, hex-1-enylene, hex-2-enylene,hex-3-enylene and hex-4-enylene groups. Thus, x is preferably 3 or 4,and y is preferably 3 or 4. Similarly, the alkylene or alkenylene grouprepresented by R^(c) is preferably a trimethylene or tetramethylenegroup. Where R^(b) is a trimethylene group, R^(c) is a tetramethylenegroup, and vice versa. Most preferably, R^(b) is a trimethylene groupand R^(c) is a tetramethylene group.

The various groups R¹, R⁴, R⁵, R⁶ and R⁷ may be lower alkyl or arylgroups which may be unsubstituted or may be substituted by at least oneof substituents a, defined above. The lower alkyl groups preferably havefrom 1 to 6 carbon atoms, and examples include the methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, hexyl and isohexyl groups, of which the methyl andethyl groups are preferred, the methyl group being most preferred. Thearyl groups are carbocyclic aromatic groups which preferably have from 6to 10 ring carbon atoms, and more preferably have 6 or 10 ring carbonatoms, for example the phenyl, 1-naphthyl and 2-naphthyl groups, ofwhich the phenyl group is preferred. Alternatively, any of these groupsmay be substituted by one or more of substituents α.

Examples of substituents α include:

-   -   halogen atoms for example chlorine, fluorine or bromine atoms;    -   amino groups;    -   alkylamino groups, in which the alkyl part preferably has from 1        to 6 carbon atoms, for example the methylamino, ethylamino,        propylamino, butyl amino, t-butylamino, pentylamino and        hexylamino groups;    -   dialkylamino groups, in which the alkyl part preferably has from        1 to 6 carbon atoms, for example the dimethylamino,        diethylamino, methylethylamino, dipropylamino, dibutylamino,        dipentylamino and dihexylamino groups;    -   cyano groups;    -   hydroxy groups;    -   alkyl groups (except when the substituted group is alkyl), for        example as exemplified above in relation to R¹ etc.;    -   aryl groups, for example as exemplified above in relation to R¹        etc.;    -   carbamoyl groups;    -   alkylcarbamoyl groups, in which the alkyl part preferably has        from 1 to 6 carbon atoms, for example the methylcarbamoyl,        ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl,        t-butylcarbamoyl, pentylcarbamoyl and hexylcarbamoyl groups; and    -   dialkylcarbamoyl groups, in which the alkyl part preferably has        from 1 to 6 carbon atoms, for example the dimethylcarbamoyl,        diethylcarbamoyl, methylethylcarbamoyl, dipropylcarbamoyl,        dibutylcarbamoyl, dipentylcarbamoyl and dihexylcarbamoyl groups.

Examples of such substituted groups include: halogen-substituted methylgroups, preferably having three halogen atoms, such as thetrichloromethyl and trifluoromethyl groups; halogen-substituted phenylgroups, such as the o-, m- and p-chlorophenyl, o-, m- andp-fluorophenyl, o-, m- and p-bromophenyl, 2,3-dichlorophenyl,2,3-difluorophenyl, 3,4-dichlorophenyl, 3,4-difluorophenyl,2,4,6-trichlorophenyl and 2,4,6-trifluorophenyl groups;amino-substituted alkyl groups, such as the aminomethyl, 2-aminoethyl,3-aminopropyl and 4-aminobutyl groups; alkylamino-substituted alkylgroups (in which the alkyl part of the alkylamino group preferably hasfrom 1 to 4 carbon atoms), such as the methylaminomethyl,2-methylaminoethyl, 3-methylaminopropyl, 4-methylaminobutyl,ethylaminomethyl, 2-ethylaminoethyl, 3-ethylaminopropyl,4-ethylaminobutyl, propylaminomethyl, 2-propylaminoethyl,3-propylaminopropyl, 4-propylaminobutyl, butylaminomethyl,2-butylaminoethyl, 3-butylaminopropyl and 4-butylaminobutyl groups;dialkylamino-substituted alkyl groups (in which each alkyl part of thedialkylamino group preferably has from 1 to 4 carbon atoms), such as theN,N-dimethylaminomethyl, 2-N,N-dimethylaminoethyl,3-N,N-dimethylaminopropyl, 4-N,N-dimethylaminobutyl,N,N-diethylaminomethyl, 2-N,N-diethylaminoethyl,3-N,N-diethylaminopropyl, 4-N,N-ethylaminobutyl, N,N-propylaminomethyl,2-N,N-propylaminoethyl, 3-N,N-propylaminopropyl, 4-N,N-propylaminobutyl,N,N-butylaminomethyl, 2-N,N-butylaminoethyl, 3-N,N-butylaminopropyl and4N,N-butylaminobutyl groups; aryl-(particularly phenyl or naphthyl)substituted alkyl groups, such as the benzyl, phenethyl, 3-phenylpropylor 4-phenylbutyl groups; carbamoyl-substituted alkyl groups, such as thecarbamoylmethyl, 2-carbamoylethyl, 3-carbamoylpropyl and4-carbamoylbutyl groups; alkylcarbamoyl-substituted alkyl groups (inwhich the alkyl part of the alkylcarbamoyl group preferably has from 1to 4 carbon atoms), such as the methylcarbamoylmethyl,2-methylcarbamoylethyl, 3-methylcarbamoylpropyl, 4-methylcarbamoylbutyl,ethylcarbamoylmethyl, 2-ethylcarbamoylethyl, 3-ethylcarbamoylpropyl,4-ethylcarbamoylbutyl, propylcarbamoylmethyl, 2-propylcarbamoylethyl,3-propylcarbamoylpropyl, 4-propylcarbamoylbutyl, butylcarbamoylmethyl,2-butylcarbamoylethyl, 3-butylcarbamoylpropyl and 4-butylcarbamoylbutylgroups; dialkylcarbamoyl-substituted alkyl groups (in which each alkylpart of the dialkylcarbamoyl group preferably has from 1 to 4 carbonatoms), such as the N,N-dimethylcarbamoylmethyl,2-N,N-dimethylcarbamoylethyl, 3-N,N-dimethylcarbamoylpropyl,4-N,N-dimethylcarbamoylbutyl, N,N-diethylcarbamoylmethyl,2-N,N-diethylcarbamoylethyl, 3-N,N-diethylcarbamoylpropyl,4-N,N-ethylcarbamoylbutyl, N,N-propylcarbamoylmethyl,2-N,N-propylcarbamoylethyl, 3-N,N-propylcarbamoylpropyl,4-N,N-propylcarbamoylbutyl, N,N-butylcarbamoylmethyl,2-N,N-butylcarbamoylethyl, 3-N,N-butylcarbanoylpropyl and4-N,N-butylcarbamoylbutyl groups; carboxy-substituted alkyl groups, suchas the carboxymethyl, 2-carboxyethyl, 3-carboxypropyl and 4-carboxybutylgroups and esters thereof; and o-, m- and p-aminophenyl,methylaminophenyl, ethylaminophenyl, propylaminophenyl,butylaminophenyl, N,N-dimethylaminophenyl, N,N-diethylaminophenyl,N,N-dipropylaminophenyl, N,N-dibutylaminophenyl, biphenylyl,carbamoylpbenyl, methylcarbamoylphenyl, ethylcarbamoylphenyl,propylcarbamoylphenyl, butylcarbamoylphenyl,N,N-dimethylcarbamoylphenyl, N,N-diethylcarbamoylphenyl,N,N-dipropylcarbamoylphenyl, N,N-dibutylcarbamoylphenyl andcarboxyphenyl groups and esters of the carboxyphenyl groups.

Examples of ester groups include:

alkyl groups having from 1 to 20 carbon atoms, more preferably from 1 to6 carbon atoms, such as those exemplified above and higher alkyl groupsas are well known in the art, such as the heptyl, octyl, nonyl, decyl,dodecyl, tridecyl, pentadecyl, octadecyl, nonadecyl and icosyl groups,but most preferably the methyl, ethyl and t-butyl groups;

cycloalkyl groups having from 3 to 7 carbon atoms, for example thecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl groups;

aralkyl groups, in which the alkyl part has from 1 to 3 carbon atoms andthe aryl part is a carbocyclic aromatic group having from 6 to 14 carbonatoms, which may be substituted or unsubstituted and, if substituted,has at least one of substituents a defined and exemplified above,although the unsubstituted groups are preferred; examples of sucharalkyl groups include the benzyl, phenethyl, 1-phenylethyl,3-phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl,2-(1-naphthyl)ethyl, 2-(2-naphthyl)ethyl, benzhydryl (i.e.diphenylmethyl), triphenylmethyl, bis(o-nitrophenyl)-methyl,9-anthrylmethyl, 2,4,6-trimethylbenzyl, 4-bromobenzyl, 2-nitrobenzyl,4-nitrobenzyl, 3-nitrobenzyl, 4-methoxybenzyl and piperonyl groups;

alkenyl groups having from 2 to 6 carbon atoms, such as the vinyl,allyl, 2-methylallyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl,3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl,2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl groups, of which thevinyl, allyl, 2-methylallyl, 1-propenyl, isopropenyl and butenyl groupsare preferred, the allyl and 2-methylallyl groups being most preferred.

halogenated alkyl groups having from 1 to 6, preferably from 1 to 4,carbon atoms, in which the alkyl part is as defined and exemplified inrelation to the alkyl groups above, and the halogen atom is chlorine,fluorine, bromine or iodine, such as the 2,2,2-trichloroethyl,2-haloethyl (e.g. 2-chloroethyl, 2-fluoroethyl, 2-bromoethyl or2-iodoethyl), 2,2-dibromoethyl and 2,2,2-tribromoethyl groups;

substituted silylalkyl groups, in which the alkyl part is as defined andexemplified above, and the silyl group has up to 3 substituents selectedfrom alkyl groups having from 1 to 6 carbon atoms and phenyl groupswhich are unsubstituted or have at least one substituent selected fromsubstituents a defined and exemplified above, for example a2-trimethylsilylethyl group;

phenyl groups, in which the phenyl group is unsubstituted orsubstituted, preferably with at least one alkyl group having from 1 to 4carbon atoms or acylamino group, for example the phenyl, tolyl andbenzamidophenyl groups;

phenacyl groups, which may be unsubstituted or have at least one ofsubstituents α defined and exemplified above, for example the phenacylgroup itself or the p-bromo-phenacyl group;

cyclic and acyclic terpenyl groups, for example the geranyl, neryl,linalyl, phytyl, menthyl (especially m- and p-menthyl), thujyl, caryl,pinanyl, bomyl, notcaryl, norpinanyl, norbomyl, menthenyl, camphenyl andnorbornenyl groups;

alkoxymethyl groups, in which the alkoxy part has from 1 to 6,preferably from 1 to 4, carbon atoms and may itself be substituted by asingle unsubstituted alkoxy group, such as the methoxymethyl,ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl andmethoxyethoxymethyl groups;

aliphatic acyloxyalkyl groups, in which the acyl group is preferably analkanoyl group and is more preferably an alkanoyl group having from 2 to6 carbon atoms, and the alkyl part has from 1 to 6, and preferably from1 to 4, carbon atoms such as the acetoxymethyl, propionyloxymethyl,butyryloxymethyl, isobutyryloxymethyl, pivaloyloxymethyl,1-pivaloyloxyethyl, 1-acetoxyethyl, 1-isobutyryloxyethyl,1-pivaloyloxypropyl, 2-methyl-1-pivaloyloxypropyl, 2-pivaloyloxypropyl,1-isobutyryloxyethyl, 1-isobutyryloxypropyl, 1-acetoxypropyl,1-acetoxy-2-methylpropyl, 1-propionyloxyethyl, 1-propionyloxypropyl,2-acetoxypropyl and 1-butyryloxyethyl groups;

cycloalkyl-substituted aliphatic acyloxyalkyl groups, in which the acylgroup is preferably an alkanoyl group and is more preferably an alkanoylgroup having from 2 to 6 carbon atoms, the cycloalkyl substituent hasfrom 3 to 7 carbon atoms, and the alkyl part has from 1 to 6, preferablyfrom 1 to 4, carbon atoms, such as the (cyclohexylacetoxy)methyl,1-(cyclohexylacetoxy)ethyl, 1-(cyclohexylacetoxy)propyl,2-methyl-1-(cyclohexylacetoxy)propyl, (cyclopentylacetoxy)methyl,1-(cyclopentylacetoxy)ethyl, 1-(cyclopentylacetoxy)propyl and2-methyl-1-(cyclopentylacetoxy)propyl groups;

alkoxycarbonyloxyalkyl groups, especially 1-(alkoxycarbonyloxy)ethylgroups, in which the alkoxy part has from 1 to 10, preferably from 1 to6, and more preferably from 1 to 4, carbon atoms, and the alkyl part hasfrom 1 to 6, preferably from 1 to 4, carbon atoms, such as the1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl,1-propoxycarbonyloxyethyl, 1-isopropoxycarbonyloxyethyl,1-butoxycarbonyloxyethyl, 1-isobutoxycarbonyloxyethyl,1-sec-butoxycarbonyloxyethyl, 1-t-butoxycarbonyloxyethyl,1-(1-ethylpropoxycarbonyloxy)ethyl and1-(1,1-dipropylbutoxycarbonyloxy)ethyl groups, and otheralkoxycarbonylalkyl groups, in which both the alkoxy and alkyl groupshave from 1 to 6, preferably from 1 to 4, carbon atoms, such as the2-methyl-1-(isopropoxycarbonyloxy)propyl,2-(isopropoxycarbonyloxy)propyl, isopropoxycarbonyloxymethyl,t-butoxycarbonyloxymethyl, methoxycarbonyloxymethyl andethoxycarbonyloxymethyl groups;

cycloalkylcarbonyloxyalkyl and cycloalkyloxycarbonyloxyalkyl groups, inwhich the cycloalkyl group has from 3 to 10, preferably from 3 to 7,carbon atoms, is mono- or poly-cyclic and is optionally substituted byat least one (and preferably only one) alkyl group having from 1 to 4carbon atoms (e.g. selected from those alkyl groups exemplified above)and the alkyl part has from 1 to 6, more preferably from 1 to 4, carbonatoms (e.g. selected from those alkyl groups exemplified above) and ismost preferably methyl, ethyl or propyl, for example the1-methylcyclohexylcarbonyloxymethyl,1-methylcyclohexyloxycarbonyloxymethyl, cyclopentyloxycarbonyloxymethyl,cyclopentylcarbonyloxymethyl, 1-cyclohexyloxycarbonyloxyethyl,1-cyclohexylcarbonyloxyethyl, 1-cyclopentyloxycarbonyloxyethyl,1-cyclopentylcarbonyloxyethyl, 1-cycloheptyloxycarbonyloxyethyl,1-cycloheptylcarbonyloxyethyl, 1-methylcyclopentylcarbonyloxymethyl,1-methylcyclopentyloxycarbonyloxymethyl,2-methyl-1-(1-methylcyclohexylcarbonyloxy)propyl,1-(1-methylcyclohexylcarbonyloxy)propyl,2-(1-methylcyclohexylcarbonyloxy)propyl,1-(cyclohexylcarbonyloxy)propyl, 2-cyclohexylcarbonyloxy)propyl,2-methyl-1-(1-methylcyclopentylcarbonyloxy)propyl,1-(1-methylcyclopentylcarbonyloxy)propyl,2-(1-methylcyclopentylcarbonyloxy)propyl,1-(cyclopentylcarzonyloxy)propyl, 2-(cyclopentylcarbonyloxy)propyl,1-(1-methylcyclopentylcarbonyloxy)ethyl,1-(1-methylcyclopentylcarbonyloxy)propyl, adamantyloxycarbonyloxymethyl,adamantylcarbonyloxymethyl, 1-adamantyloxycarbonyloxyethyl and1-adamantylcarbonyloxyethyl groups;

cycloalkylalkoxycarbonyloxyalkyl groups in which the alkoxy group has asingle cycloalkyl substituent, the cycloalkyl substituent having from 3to 10, preferably from 3 to 7, carbon atoms and mono- or poly-cyclic,for example the cyclopropylmethoxycarbonyloxymethyl,cyclobutylmethoxycarbonyloxymethyl, cyclopentylmethoxycarbonyloxymethyl,cyclohexylmethoxycarbonyloxymethyl,1-(cyclopropylmethoxycarbonyloxy)ethyl,1-(cyclobutylmethoxycarbonyloxy)ethyl,1-(cyclopentylmethoxycarbonyloxy)ethyl and1-(cyclohexylmethoxycarbonyloxy)ethyl groups;

terpenylcarbonyloxyalkyl and terpenyloxycarbonyloxyalkyl groups, inwhich the terpenyl group is as exemplified above, and is preferably acyclic terpenyl group, for example the 1-(menthyloxycarbonyloxy)ethyl,1-(menthylcarbonyloxy)ethyl, menthyloxycarbonyloxym ethyl,menthylcarbonyloxymethyl, 1-(3-pinanyloxycarbonyloxy)ethyl,1-(3-pinanylcarbonyloxy)ethyl, 3-pinanyloxycarbonyloxymethyl and3-pinanylcarbonyloxymethyl groups;

5-alkyl or 5-phenyl [which may be substituted by at least one ofsubstituents α, defined and exemplified above](2-oxo-1,3-dioxolen-4-yl)alkyl groups in which each alkyl group (whichmay be the same or different) has from 1 to 6, preferably from 1 to 4,carbon atoms, for example the (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl,(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl,(5-isopropyl-2-oxo-1,3-dioxolen-4-yl)-methyl,(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and1-(5-methyl-2-oxo-1,3-dioxolen-4-yl)ethyl groups; and

other groups, especially groups which are easily removed in vivo such asthe phthalidyl, indanyl and2-oxo-4,5,6,7-tetrahydro-1,3-benzodioxolen-4-yl groups.

Of the above groups, we especially prefer those groups which can beremoved easily in vivo, and most preferably the aliphatic acyloxyalkylgroups, alkoxycarbonyloxyalkyl groups, cycloalkylcarbonyloxyalkylgroups, phthalidyl groups and (5-substituted2-oxo-1,3-dioxolen-4-yl)methyl groups.

However, we prefer that R¹, R⁴, R⁵, R⁶ and R⁷ are all hydrogen.

It is generally preferred that the group Z is not present, i.e. n is 0,but where it is present then it is preferably that it corresponds to theresidue of an aromatic, and preferably hydrophobic aromatic, amino acid,more preferably an α-amino acid, such as histidine, phenylalanine,tyrosine, tryptophan or phenylglycine, of which phenylalanine ortyrosine are most preferred.

R^(c) is a lower alkylene group optionally substituted by 1 or 2 alkyl,preferably methyl, groups. Such a lower alkylene group has 3 or 4 carbonatoms in a straight chain and is optionally substituted by 1 or 2 alkyl,preferably methyl, groups. Examples of such groups include themethylene, ethylene, methylethylene, 1-, 2- or 3-methyl-trimethylene,trimethylene, propylene, tetramethylene, pentamethylene andhexamethylene groups, of which the trimethylene and tetramethylenegroups are generally preferred.

Preferred compounds of the present invention are the following Compoundsof formula A to D:

Compound of Formula A:

Compound of Formula B:

Compound of Formula C:

Compound of Formula D:

Compound of Formula E:

Compound of Formula F:

Compound of Formula G:

Compound of Formula H:

Compound of Formula I:

Of these, the Compounds of formula A, D, E, F, G, H and I are especiallypreferred, the Compounds of formula A and D being more preferred, andthe Compound of formula A being most preferred.

The compounds of the present invention may be prepared by a variety ofprocesses which, in themselves, are well-known in the art.Alternatively, they may be prepared by the following procedure:

Wang resin (0.03 mmol) is swollen in anhydrous tetrahydrofuran (1.0 ml)and carbonyl diimidazole (4 equivalents, 0.12 mmol) is addedportion-wise. The resulting mixture is stirred at ambient temperaturefor 16 hours and then filtered, after which it is washed withtetrahydrofuran, ethanol and dichloromethane. The resin is then dried invacuo.

The resin is re-swollen in anhydrous dichloromethane (1.0 ml) and1,3-diaminopropane (10 equivalents, 0.3 mmol) is added portion-wise. Theresulting mixture is stirred for 2 hours and then filtered, after whichit is washed (dimethylformamide, methanol, dichloromethane) and thendried in vacuo.

The resin is again re-swollen in anhydrous dichloromethane (1.0 ml) and2,6-lutidine (5 equivalents, 0.15 mmol) is added, followed by thecareful addition of 2,4-dinitrobenzenesulfonyl chloride (4 equivalents,0.12 mmol). The mixture is stirred under an inert atmosphere for 2 hoursand then washed (dimethylformamide, methanol, dichloromethane) and driedin vacuo.

The resulting resin is then swollen in anhydrous tetrahydrofuran (1.0ml) and triphenylphosphine (4 equivalents, 0.12 mmol), Dde-protectedaminoalcohol (4 equivalents, 0.12 mmol) are added and dissolved withstirring. Diethylazodicarboxylate (4 equivalents, 0.12 mmol) is addeddropwise and the mixture stirred for 12 hours and then filtered andwashed (dimethylformamide, methanol, dichloromethane), after which it isdried in vacuo.

The resin is then swollen in dichloromethane (1.0 ml) and propylamine (5equivalents, 0.15 mmol) is added and the mixture is stirred for 1 hour,after which it is filtered and washed (dimethylformamide, methanol,dichloromethane) and then dried in vacuo.

The resin is again swollen in dichloromethane (1.0 ml) and di-t-butyldicarbonate (10 equivalents, 0.3 mmol) and N,N-dimethylaminopyridine (5mol %, 0.0015 mmol) are added. The is then mixture stirred for 16 hours.The resin is then filtered and washed (dimethylformamide, methanol,dichloromethane) and then dried in vacuo.

The resin is then stirred in 2% hydrazine hydrate/dimethylformamide (1.0ml) for 1 hour then washed (dimethylformamide, methanol,dichloromethane) and dried in vacuo.

Fmoc AA (4 equivalents, 0.12 mmol), TBTU (4 equivalents, 0.12 mmol), anddiisopropylethylamine (8 equivalents, 0.48 mmol) are dissolved inanhydrous dimethylformamide (1.0 ml) and the mixture added to the resin.The whole is then stirred for 12 hours and then filtered and washed(dimethylformamide, methanol, dichloromethane) and dried in vacuo.

To the resin is added 20% piperidine/dimethylformamide (1.0 ml) and themixture is stirred for 0.5 hour and then filtered and washed(dimethylformamide, methanol, dichloromethane), after which it is driedin vacuo.

Boc AA (4 equivalents, 0.12 mmol), TBTU (4 equivalents, 0.12 mmol), anddiisopropylethylamine (8 equivalents, 0.48 mmol) are dissolved indimethylformamide (1.0 ml) and the mixture is added to the resin. Thewhole is then stirred for 12 hours and then filtered and washed(dimethylformamide, methanol, dichloromethane), after which it is driedin vacuo.

50% TFA/45% dichloromethane/2.5% H₂O/2.5% triisopropylsilane (1.0 ml) isadded to the resin and the mixture is stirred for 1 hour. The resin isfiltered and washed with dichloromethane (1.0 ml) and the filtrate isconcentrated in vacuo. The resulting viscous yellow oil is trituratedwith anhydrous diethyl ether (3×2 ml) to yield the required compound.

Preparation of the compounds of the invention, as well asneuroprotective activity is illustrated in the accompanying non-limitingexamples. In these examples, the following abbreviations are used:

-   -   Arg arginine;    -   Boc t-butoxycarbonyl;    -   DIC di-isopropylcarbodiimide;    -   EDT ethane-1,2-diol;    -   Fmoc N-fluorenylmethoxycarbonyl;    -   HOBt hydroxybenzotriazole;    -   Lys lysine;    -   ODS octadecylsilane    -   Orn ornithine;    -   Phe phenylalanine;    -   Pmc N^(G)-2,2,5,7,8-pentamethylchroman-6-ylsulphonyl;    -   RP-HPLC reverse phase high performance liquid chromatography;    -   TFA trifluoroacetic acid;

COMPOUND SYNTHESIS EXAMPLE 1 N¹-L-Arginylspermidine [Compound of FormulaA]

0.152 g ofN¹-Fluorenylmethoxycarbonyl-N⁴-(4′-benzoyloxycarbonyl-(1′-phenoxy)ethanoamidoresin)-N⁸-t-butoxycarbonylspermidine was treated with 5 ml of a 20% v/vsolution of piperidine in dimethylformamide. The resin was filtered, andthen treated again with 5 ml of a 20% v/v solution of piperidine indimethylformamide for a further 30 minutes. At the end of this time, theresin was filtered and washed, in that order, with 10 ml ofdimethylformamide, 5 ml of methanol and finally twice, each time with 10ml of methylene chloride. Fmoc-Arg(Pmc)OH (0.1027 g, 0.154 mmol) wasdissolved in methylene chloride (9 ml), and then HOBt (0.021 g, 0.155mmol) was added. After 10 minutes at room temperature,N⁴-(4′-Benzoyloxycarbonyl (1′-phenoxy) ethanoamidoresin)-N⁸-t-butoxycarbonylspermidine (0.1032 g, 0.031 mmol) was addedfollowed by DIC (24 ml, 0.155 mmol). The mixture was gently stirred atroom temperature for 20 hours. Following a negative ninhydrin test theresin was filtered and washed with methylene chloride (1×10 ml),methanol (1×5 ml), methylene chloride (2×10 ml) then dried under vacuum.Fmoc removal was carried out as above.N¹-Arg(Pmc)-N⁴-(4′-Benzoyloxycarbonyl-(1′-phenoxy)ethanoamidoresin)-N⁸-t-butoxycarbonylspermidine was deprotected/cleaved usingTFA-phenol-water-triisopropylsilane-ethane-1,2-dithiol (EDT)(81.5:5:5:1:2.5 by volume; 2.5 ml) for 5 hours at room temperature. Theresin was removed by filtration through a Pasteur pipette containing atight plug of glass wool and washed with methylene chloride (4×4 ml).The solvent was removed in vacuo, the residue dissolved in CH₃CN (1 ml)and poured into cold diethyl ether (25 ml) to give a white precipitatewhich was separated by centrifugation. The supernatant was decanted andthe solid resuspended in diethyl ether (25 ml). The solid was againseparated by centrifugation and the procedure repeated twice. Theproduct was dissolved in water before freeze-drying. The product (19.2mg) was analysed and purified by RP-HPLC (ODS, eluting isocraticallywith water/0.1% TFA).

EXAMPLE 2 Compounds B, C, Z¹, Z² and Z³

These Compounds were prepared in an analogous manner usingFmoc-L-Lys(t-butoxycarbonyl), Fmoc-L-Orn(t-butoxycarbonyl),Fmoc-D-Arg(Pmc), Fmoc-D-Lys(t-butoxycarbonyl) andFmoc-D-Orn(t-butoxycarbonyl), respectively.

Compound Analysis N-L-Arginylspermidine (Compound of Formula A)

δH (300 MHz, D₂O): 3.86 (1H, t, J-6.6, Arg alpha-CH), 3.28–3.02 (4H, m),2.95–2.78 (6H, m), 1.98–1.70 (4H, m), 1.68–1.40 (6H, m)

δC (75 MHz, D₂O): 173.1 (COOH), 159.6 (NH═C(NH-2)NH), 55.6 (CH), 49.6(CH₂), 47.8 (CH₂), 42.9 (CH₂), 41.4 (CH₂), 39.1 (CH₂), 30.8 (CH₂), 28 1(CH₂), 26.5 (CH₂), 26.3 (CH₂), 25.4 (CH₂)

M/Z: (ES+) 302.3 (M+H)⁺, 416.3 (M+H+TFA)⁺.

N₁-D-Arginylspermidine (Compound Z¹)

δH (360 MHz, D₂O): 3.78 (1H, t, J-6.5 Arg alpha-CH), 3.32–3.04 (4H, m),3.03–2.83 (6H, m), 1.87–1.69 (4H, m), 1.68–1.55 (4H, m), 1.54–1.42 (2H,m)

δC (95 MHz, D₂O): 53.8 (CH), 47.7 (CH₂), 45.9 (CH₂), 41.1 (CH₂), 39.5(CH₂), 37.2 (CH₂), 29.0 (CH₂), 26.2 (CH₂), 24.6 (CH₂), 24 4 (CH₂), 23 5(CH₂)

M/Z: (ES+) 302.3 (M+H)⁺, 416.3 (M+H+TFA)⁺.

N¹-L-Lysinylspermidine [Compound of Formula B]

δH (360 MHz, D₂O): 3.84 (1H, t, J-6.6, Lys alpha-CH), 3.23 (2H, aft, J7.5), 3.09–2.80 (8H, m), 1.89–1.73 (4H, m), 1.72–1.49 (6H, m), 1.44–1.26(2H, m);

M/Z: (ES+) 274.3 (M+H)⁺, 410.3 (M+Na+TFA)⁺.

N¹-D-Lysinylspermidine (Compound Z²)

δH (360 MHz, D₂O): 3.84 (1H, t, J-6.5, Lys alpha-CH), 3.23 (2H, aft, J7.5), 3.09–2.84 (8H, m), 1.90–1.74 (4H, m), 1.73–1.50 (6H, m), 1.40–1.27(2H, m)

M/Z: (ES+) 274.3 (M+H)⁺, 388.4 (M+H+TFA)⁺.

N¹-L-Ornithylspermidine [Compound of Formula C]

δH (360 MHz, D₂O): 3.94 (1H, t, J-6.6, Orn alpha-CH), 3.31 (2H, aft, J7.5), 3.18–2.89 (8H, m), 2.08–1.80 (4H, m), 1.78–1.52 (6H, m)

M/Z: (ES+) 260.3 (M+H)⁺, 374.3 (M+H+TFA)⁺.

N¹-D-Ornithylspermidine (Compound Z³)

δH (360 MHz, D₂O): 3.88 (1H, t, J-6.6, Orn alpha-CH), 3.23 (2H, aft, J7.5), 3.10–2.80 (8H, m), 1.98–1.78 (4H, m), 1.75–1.50 (6H, m)

M/Z: (ES+) 260.3 (M+H)⁺, 374.3 (M+H+TFA)⁺.

HPLC Analysis

The compounds of the present invention were analysed by HPLC. Theresults showed that the compounds when made by the preferred process ofthe present invention were substantially free of original reactants.

EXAMPLE 3 Arginine-L-phenylalanine-spermidine: Compound of Formula G

Wang resin (0.03 mmol, 50 mg) was swollen in anhydrous tetrahydrofuran(1.0 ml) and carbonyl diimidazole (4 equivalents, 0.12 mmol, 19 mg) wasadded. The resulting mixture was then stirred at ambient temperature for16 hours, after which it was filtered and washed with tetrahydrofuran,ethanol and dichloromethane. The resin was then dried in vacuo.

The resin was re-swollen in anhydrous dichloromethane (1.0 ml), and1,4-diaminobutane (10 equivalents, 0.3 mmol, 25 mg) were added. Theresulting mixture was stirred for 2 hours and then filtered and washed(dimethylformamide, methanol, dichloromethane), after which it was driedin vacuo.

The resin was again re-swollen in anhydrous dichloromethane (1.0 ml),and 2,6-lutidine (5 equivalents, 0.15 mmol, 16 mg) were added, followedby the careful addition of 2,4-dinitrobenzenesulfonyl chloride (4equivalents, 0.12 mmol, 32 mg). The mixture was stirred under an inertatmosphere for 2 hours and then washed (dimethylformamide, methanol,dichloromethane) and dried in vacuo.

The resulting resin was then swollen in anhydrous tetrahydrofuran (1.0ml) and triphenylphosphine (4 equivalents, 0.12 mmol, 32 mg).Dde-protected aminoalcohol (4 equivalents, 0.12 mmol, 29 mg) were addedand dissolved with stirring. Diethyl azodicarboxylate (4 equivalents,0.12 mmol, 21 mg) was added dropwise and the mixture was stirred for 12hours and then filtered and washed (dimethylformamide, methanol,dichloromethane). It was then dried in vacuo.

The resin was then swollen in dichloromethane (1.0 ml), and propylamine(5 equivalents, 0.15 mmol, 13 mg) was added. The mixture was thenstirred for 1 hour after which it was filtered and washed(dimethylformamide, methanol, dichloromethane) and then dried in vacuo.

The resin was again swollen in dichloromethane (1.0 ml), and dibutyldicarbonate (10 equivalents, 0.3 mmol, 33 mg) andN,N-dimethylaminopyridine (5 mol %, 0.0015 mmol, 0.2 mg) were added, andthe mixture was stirred for 16 hours. The resin was then filtered andwashed (dimethylformamide, methanol, dichloromethane), and then dried invacuo.

The resin was then stirred in 2% hydrazine hydrate/dimethylformamide(1.0 ml) for 1 hour and then washed (dimethylformamide, methanol,dichloromethane), after which it was dried in vacuo.

Fmoc-Phe-OH (4 equivalents, 0.12 mmol, 46 mg), TBTU (4 equivalents, 0.12mmol, 39 mg) and diisopropylethylamine (8% 0.48 mmol, 62 mg) weredissolved in anhydrous dimethylformamide (1.0 ml), and the mixture wasadded to the resin. The whole was then stirred for 12 hours, and thenfiltered and washed (dimethylformamide, methanol, dichloromethane) anddried in vacuo.

To the resin was added 20% piperidine/dimethylformamide (1.0 ml) and themixture was stirred for 0.5 hour. It was then filtered and washed(dimethylformamide, MEOH, dichloromethane) and then dried in vacuo.

Boc-Arg(Phe-OH (4 equivalents, 0.12 mmol, 63 mg), TBTU (4 equivalents,0.12 mmol, 39 mg), and diisopropylethylamine (8 equivalents, 0.48 mmol,62 mg) were dissolved in dimethylformamide (1.0 ml) and the mixture wasadded to the resin. The whole was then stirred for 12 hours and thenfiltered and washed (dimethylformamide, methanol, dichloromethane). Itwas then dried in vacuo.

50% TFA/45% dichloromethane/2.5% H₂O/2.5% triisopropylsilane (1.0 ml)was added to the resin and the mixture was stirred for 1 hour. The resinwas filtered and washed with dichloromethane (1.0 ml) and the filtratewas concentrated in vacuo. The resulting viscous yellow oil wastriturated with anhydrous diethyl ether (3×2 ml) to yield the titlecompound as its tetrakis TFA salt (19 mg, 700/o):

Analysis:

LCMS

90% (ELS detection). M/z 449 (ES⁺).

NMR:

¹H NMR was found to be in accordance with the above structure

Dde protected aminoalcohol:

Dde=N-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl

Preparation of Dde Protected Aminoalcohol

To a solution of 3-amino-1-propanol (1.5 g, 20 mmol) in ethanol wasadded 2-acetyl dimedone (1.1 equivalents, 22 mmol, 4.0 g) and themixture was heated to 50° C. for 1 hour. The resulting solution wasconcentrated in vacuo to yield a red crystalline solid that wastriturated with hexane to afford an off-white solid (4.74 g, 95%)

EXAMPLE 4 Compound of Formula D

A PTFE 2 ml syringe was filled withN¹-Fluorenylmethoxycarbonyl-N⁴-(4′-benzoyloxycarbonyl(1′-phenoxy)ethanoamidoresin)-N⁸-Bocspermidine (≈31 mg, 0.263 mmol/g) and treated 3 times with20% piperidine in dimethylformamide (2 ml) for 30 minutes, followed bywashing with dimethylformamide (2×2 ml) and CH₂Cl₂ (4×2 ml).

The resulting primary amine was coupled to Fmoc-Phe) using 5 equivalents(0.041 mmol) of the Fmoc-carbamoyl acid and DIC/HOBt activation inCH₂Cl₂/dimethylformamide (1 ml/1 drop). After 4 hours with occasionalstirring, ninhydrin tests indicated that the couplings was complete.After treatment with 20% piperidine in dimethylformamide (2 ml, 2×30 mn)and washing with dimethylformamide (2×2 ml) and CH₂Cl₂ (4×2 ml)Di(Boc)-protected guanidino carboxylic acid was coupled to the sample.Coupling was achieved using 3 equivalents (0.025 mmol) of the carboxylicacid with DIC/HOBt activation in CH₂Cl₂/dimethylformamide (1 ml/1 drop).After 5 hours with occasional stirring, ninhydrin tests showed that thecoupling was complete.

After washing with dimethylformamide (2×2 ml) and CH₂Cl₂ (4×2 ml), thecompound was deprotected-cleaved from the solid support, the resin beingpre-swollen in CH₂Cl₂ (1 ml) prior to treatment with TFA-H₂O (95:5, 0.4ml) for 1.5 hours.

The resin sample was washed with TFA-CH₂Cl₂ (1:1, 2 ml) and then thewash filtered into a vial. The solvent was reduced in vacuo and theresidue was dissolved in water, frozen and lyophilised. The compound wasanalysed by ES MS and gave the desired molecular ion as the major peak.

M/Z: (ES⁺) 448.4

EXAMPLE 5 Protocol For Studying Hypoxic Neuronal Damage

Hypoxic neuronal damage was studied using organotypic hippocampal slicecultures [Pringle A. K. et al. (1996 Stroke 27 2124–2130)].

Cultures were prepared according to the method of Stoppini et al (1991J. Neurosci. Meth. 37 173–182) from 8–10 day old Wistar rat pups(Bioresources Unit, University of Southampton). Cultures were maintainedin vitro for 14 days (37° C., 5% CO₂) during which the medium (50%minimum essential medium (MEM), 25% Hank's balanced salt solution(HBSS), 25% heat-inactivated horse serum, supplemented with 1 mMglutamine, 5 mg/ml glucose and 1.5% fungizone) was changed every 3 days.Hypoxia was induced by replacing culture medium with serum-free (SF)medium (75% MEM, 25% HBSS, 1 mM glutamine, 5 mg/ml glucose, 1.5%fungizone) saturated with 95% N₂/5% CO₂ (and thus oxygen-free), andplacing cultures in an air-tight chamber in which the atmosphere wasalso saturated with N₂/CO₂. After 180 minutes hypoxia, cultures werereplated in normoxic SF medium and replaced in the incubator for 24hours. Compounds were added to cultures either pre-, during andpost-hypoxia (herein abbreviated to “pdp”) or just in the post-hypoxicrecovery period (“post”) [Johnson, T. D. (1996 Trends Pharmacol. Sci.22–27)]. Cell damage was evaluated using the fluorescent exclusion dyepropidium iodide (PI, 5 μg/ml) which is normally excluded from healthycells, but enters cells with damaged plasma membranes and becomes highlyfluorescent when bound to DNA. Neuronal cell damage was quantified usingthe “NIH Image 1.55” software. Briefly, the area of the CA1, CA3 anddentate gyrus (DG) cell layers was measured from a transmission image.Twenty-four hours after the commencement of hypoxia, a fluorescenceimage was captured using a standard Leica inverted fluorescencemicroscope fitted with a rhodamine filter set. The area of PIfluorescence above background in the neuronal cell layers was determinedusing the density slice function of Image. Cellular damage is expressedas the percentage area of the cell body layers in which PI fluorescencewas detectable. After imaging, cultures were fixed overnight in 4%paraformaldehyde and stained with thionin.

Data are expressed as the mean±sem. Data from the non-drug groups waspooled before analysis. Statistical significance was determined usingone-way analysis of variance (ANOVA) followed by post-hoc non-pairedStudent's t-tests. As only cells within the CA1 region were susceptibleto hypoxia-induced damage, all of the pharmacological data wascalculated for this region alone.

Protocol for Studying NMDA Receptor-Mediated Neurotoxicity

Organotypic hippocampal slice cultures were prepared and maintained asdescribed above. NMDA was prepared as a 50 mM stock solution indistilled water, and diluted as required in SF medium. Neurotoxicity wasinduced by placing cultures in SF medium containing either 10 μM or 30μM NMDA for 180 minutes. After this time, cultures were replated inSF-medium and maintained for 24 hours in the incubator. Either 300 μML-ArgSp or vehicle (SF medium) was added to the culture medium pre-,during and post-NMDA exposure. Throughout the duration of theexperiment, 5 μg/ml PI was included in the medium. After 24 hours,neuronal damage was determined by PI fluorescence imaging and quantifiedas described previously.

Blood Flow Studies

Adult male Wistar rats (250–300 g) were initially anaesthetised with 4%halothane and subsequently anaesthesia was maintained with 1.5%halothane mixed in 7% N₂O, in O₂. The femoral artery was cannulated forcontinuous blood pressure recording. The femoral vein was alsocannulated to allow injection of the compound. Animals were allowed15–30 minutes to stabilise and were then injected with 0.25–0.3 ml of a1 mg/ml solution of L-ArgSp. Following injection of the compound, ratswere continuously monitored for 60 minutes. After this time, anaesthesiawas terminated and rats allowed to waken. In these studies, measurementwas taken of the mean arterial blood pressure (MABP) induced followingan intravenous injection of 1 mg/kg L-ArgSp into the femoral vein ofanaesthetised male Wistar rats. MABP was calculated immediately prior toinjection of L-ArgSp (pre-injection) and 30 seconds and 10 minutespost-injection.

Data are presented as mean±sem of four observations.

Global Forebrain Ischaemia

Animals were anaesthetised as described above, and a thermistor insertedinto the left temporal muscle for recording of body temperature. Adorsal midline skin incision was made in the neck and usingmicrosurgery, the vertebral arteries were identified and occluded usinga monopolar electrode at the level of C 1. The incision was closed,animals allowed to recover from anaesthesia and returned to their cagesfor 24 hours. After this time, animals were re-anaesthetised and thecommon carotid arteries (CCAs) exposed. Animals were divided into twogroups. Group 1 received 0.25–0.3 ml of a 1 mg/ml solution of L-ArgSp(final dose 1 mg/kg) while group 2 received 0.25–0.3 ml of steriledistilled water. Samples were prepared independently and randomisedprior to injection. Animals were injected 15 minutes prior to occlusionof the CCAs with microvascular clips for 15 minutes. After this time,the skin was closed, and animals allowed to recover. After 24 hoursanimals were terminally anaesthetised and transcardially perfused with1% paraformaldehyde and the brains removed and processed for histology.A blinded observer determined the number of live and dead neurones inthe CA1, CA3 fields of the pyramidal cell layer, and the dentate gyrusgranule cell layer from haematoxylin and eosin stained coronal sections.

Results

(i) Control Procedures

After 14 days in vitro, organotypic hippocampal slice cultures retainedmuch of the structure and morphology of the in vivo hippocampus.Specifically, clearly identifiable pyramidal (CA1 and CA3/4) and dentategyrus granule cell layers were visible in thionin stained sections.Neurons appeared healthy, with large, lightly-stained nuclei surroundedby intensely-staining cytoplasm.

24 hours after 3 hours hypoxia, PI fluorescence was detectable in theCA1 region of the pyramidal cell layer (35.6±1.43% damage, n=108), withlittle (but not statistically significant) PI labelling in either theCA3 pyramidal cells (7.1±1.3%) or dentate granule cells (3.9±2.9%).Little PI fluorescence was observed in untreated controls maintained inserum-free medium for 24 hours. After thionin-staining, control cultureswere indistinguishable from untreated slices, with large, healthyappearing neurones. In contrast, in cultures exposed to 180 minuteshypoxia the CA3 pyramidal cells and dentate granule cells appearednormal, but cells in CA1 had small, darkly-staining, pyknotic nucleiwith little visible cytoplasm indicating neuronal death—as shown inFIG. 1. FIG. 1 shows PI fluorescence images of untreated control culture(A) or culture 24 hours after a 3 hours hypoxia (B). No PI fluorescenceis detectable in the untreated culture, but intense staining is presentin the CA1 pyramidal cell layer of the hypoxia-treated culture. (C+D)Corresponding thionin-stained sections of CA1 region of the culturesshown in A+B. (C) Neurones contain lightly-stained nucleus surrounded bydarkly-staining cytoplasm (white arrow). (D) In contrast, neurones inthe hypoxia-treated cultures appear as small, darkly-staining pyknoticnuclei (black arrow).

On this Figure, the scale bars are: A, B: 1 mm; C, D: 100 μm.

(ii) Effects of L-Arginylspermidine (Compound A)

After incubation with 300 μM L-ArgSp for 24 hours, no increase in PIfluorescence above baseline was observed. Addition of 300 μM L-ArgSp for30 minutes prior to hypoxia, during the hypoxic episode and the 24 hourrecovery period was completely neuroprotective. PI fluorescence wasdetectable in 0.2±0.02% of CA1 (n=12, p<0.001 vs hypoxia controls). Inthionin stained slices, the neurons were indistinguishable from those ofuntreated or control cultures.

When the addition of the L-ArgSp was delayed until immediatelypost-hypoxia, a significant neuroprotective effect was still observed.This was concentration dependent (0.3–300 μM), with the EC₅₀ lyingbetween 3 and 30 μM. The damage observed in the CA1 subfield in thesecultures was reduced (see Table 1—shown below) demonstrating thatdelaying the addition of the compound did not significantly reduce theneuroprotective efficacy.

TABLE 1 Compound n % Damage CA1 % Protection Control Hypoxia 108  35.6 ±1.43 (A) L-ArgSp (300 μM) pre 12   0.2 ± 0.02*** 99.4 (A) L-ArgSp (300μM) 16   9.9 ± 3.5*** 72.2 (Z¹) D-ArgSp (300 μM)  8 32.6 ± 4.1  8.4 (B)L-LysSp (300 μM) 16 27.0 ± 3.7* 24.2 (Z²) D-LysSp (300 μM) 14 36.3 ±3.5  0 (C) L-OrnSp (300 μM) 12 30.6 ± 3.8  14.0 (Z³) D-OrnSp (300 μM) 1136.71 ± 3.2  0 L-Arg (300 μM) 11 38.5 ± 4.0  0 D-Arg(300 μM) 10 32.7 ±7.0  8.1 Spermidine (300 μM) 12 34.2 ± 5.4  3.9

Table 1 represents the quantification of the percentage area of the CA1pyramidal cell layer in which PI fluorescence was detectable 24 hoursafter 3 hours of hypoxia (% Damage CA1). Data from all of the culturesexposed to hypoxia alone were pooled (control hypoxia). The percentageneuroprotection was calculated as the (((% damage control hypoxia−%damage drug treated)/% damage control hypoxia)*100). Data are expressedas the mean±sem, n=number of cultures, *p<0.05, **<0.01, ***p<0.001 vshypoxia control.

To determine whether both the spermidine and arginine components of theL-ArgSp were essential for the generation of the neuroprotective effect,we also assessed the effects of post-hypoxic addition of 300 μMspermidine and 300 μM L-arginine.

Neither spermidine nor L-arginine individually produced a reduction indamage (see Table 1). These data indicate that it is necessary to have acompound having the structure as defined above—such as a compoundprepared by conjugating L-arginine with spermidine—for theneuroprotective efficacy. This result is in contrast to the findings ofWO 91/00853 wherein it is claimed that spermidine directly blockscalcium conductances. With our present work, we have shown that purifiedspemidine has no neuroprotective effects in our assay. At this stage, webelieve that the difference is attributable to the fact that in WO91/00853 no purification was attempted with the spermidine and so onecan only postulate that the spermidine used was impure.

iii) Effect of Changing the Carbamoyl Acid Side Chain

When the arginine residue was replaced with the related carbamoyl acidslysine or ornithine, the neuroprotective efficacy of the resultingcompounds was less than L-ArgSp. Nevertheless, neuroprotective efficacywas still observed. Addition of 300 μM L-lysinylspermidine (L-LysSp)immediately post hypoxia produced a small but significant reduction inPI fluorescence in the CA1 region (see Table 1). Post-hypoxic additionof 300 μM L-ornithylspermidine (L-OrnSp) produced less of a significantreduction in damage.

iv) Stereospecificity of the Neuroprotective Effect

Substitution of the L-carbamoyl acids with their respectiveD-enantiomers produced a profound reduction of the neuroprotectiveefficacy of the compounds relative to the L-enantiomers, as addition of300 μM D-ArgSp, D-LysSp or D-OrnSp post-hypoxia did not result in anyobservable reduction of PI fluorescence (see Table 1 supra). Inaddition, cells of the CA1 subfield appeared with shrunken,darkly-staining, pyknotic nuclei indicating neuronal death. Theseresults clearly demonstrate that we have found that L optical activityis important for neuroprotective efficacy. Hence, highly preferredcompounds of the present invention have L optical activity. Furthermore,and in direct contrast to the teachings of WO 91/00853, we found thatsubstituting lysine for arginine (compound A and B) reduces theneuroprotective action but does not reverse it.

v) Histogram

n=108 control. n=140.3 μM, n=73 μM, n=1430 μM, n=16300 μM).

vi) L-ArgSp Does Not Prevent NMDA-mediated Neuronal Damage

Twenty-four hours after 180 minutes exposure to 10 μM NMDA, PIfluorescence was detectable in the CA1 subfield of the pyramidal celllayer, but not other areas of the cultures. Increasing the concentrationof NMDA to 30 μM produced a more severe insult, with significantneuronal damage occurring in both the CA1 and CA3 regions of thepyramidal cell layer, but with sparing of the granule cells of thedentate gyrus. Addition of 300 μM L-ArgSp to the medium throughout theexperiment did not reduce the damage produced by either 10 μM or 30 μMNMDA. Neuronal damage is expressed as the percentage area of either CA1(solid bars) or CA3 (hatched bars) in which PI fluorescence was measured24 hours after 180 minutes exposure to NMDA. (mean+sem, n=8 for eachgroup).

vii) Blood Flow Studies

The results of these studies are presented in the Table 2 presentedbelow.

TABLE 2 Time MABP (mm Hg) % Change pre-injection 79.9 ± 3.9 30 secs 72.2± 5 1 −9.6 10 minutes 79.2 ± 6.5 −0.9

The blood pressure recordings were made 60 minutes after injection,immediately before the rat was wakened, were identical to those 10minutes post-drug administration. The small reduction in MABP producedby L-ArgSp was not statistically significant. No effect on either bodytemperature or heart rate occurred in these animals followingadministration of L-ArgSp. Following wakening, no ill effects of thecompound on the animals was observed. A further five rats have beenallowed to recover for three days following administration of 1 mg/kgL-ArgSp and no long-term behavioural deficits have been observed inthese animals.

viii) L-ArgSp Reduces Neuronal Damage Following Global ForebrainIschaemia in vivo

Fifteen minutes global forebrain ischaemia is a particularly severeinsult, producing neuronal damage throughout the hippocampal formation.When assessed 24 hours after ischaemia, animals which received vehiclealone showed a neuronal loss in CA1, CA3 and the dentate gyrus withseverity being regionally dependent (CA1>CA3>DG). In animals treatedwith 1 mg/kg L-ArgSp 15 minutes prior to induction of ischaemia, theneuronal loss was significantly attenuated, particularly in theextremely vulnerable CA1 region. (**P<0.01, *P<0.05 vs vehicle-treatedcontrols; data represents mean+sem, n=5 control, n=7 L-ArgSp).

Example Discussion

Using solid phase chemistry techniques we have synthesised, amongothers, arginylspermidine. We have shown that this compound, inparticular the L-enantiomer, possesses significant neuroprotectiveefficacy, even if the addition of the compound was delayed until afterthe termination of the hypoxic episode.

The data demonstrate that the compounds of the present invention mustcomprise the above-mentioned first component linked to theabove-mentioned second component via an amide bond—such as conjugatedspermidine and L-Arg—to have neuroprotective efficacy. In this regard,no efficacy was detected using spermidine and L-Arg on their own. Oneconclusion that could, therefore, be drawn is that the action of thecompound of the present invention, in particular L-ArgSp, is mediatedthrough a receptor site which requires the presence of both the firstand the second components in the same molecule.

Substitution of L-arginine with L-lysine (L-LysSp) or ornithine(L-OrnSp) still yields active compounds. However, the activities ofthese compounds are not as great as that for L-ArgSp. This suggests thatthe guanidinium functionality is desirable for optimal activity but thatother positively charged groups can take its place relativelysuccessfully.

It is currently believed therefore that a highly preferred feature foractivity is the relative spatial positioning of the terminal positivecharge on the guanidinium or ammonium ion and the alpha-carbamoyl group.This is suggested by the relative length of the side chains and theirreduction in overall length in going from Arg to Lys to Orn and suggeststhat the compounds are capable of exhibiting a bi-functional bindingability.

Also, in a highly preferred embodiment of the present invention, itappears that the binding site appears to be stereospecific, requiringthe carbamoyl acid to be in the L-configuration for optimal activity.Both D-ArgSp and D-LysSp were inactive relative to their correspondingL-enantiomers.

The blood flow data show that the compounds of the present invention, inparticular the Compound of formula A, have a less adverse effect onblood flow than FTX.

EXAMPLE 6

The procedures of Example 5 were repeated, but using different doses ofa fresh batch of Compound A (pdp). The results are shown in thefollowing Table 3.

TABLE 3 Compound n % Damage CA1 % Protection None (Control Hypoxia) 50 23.9 ± 2.7   0.3 μM Compound A pdp 8 20.2 ± 8.4  15.5  1 μM Compound Apdp 8 20.1 ± 8.2  15.9  3 μM Compound A pdp 15   6.5 ± 3.0** 72.8  10 μMCompound A pdp 7  8.8 ± 3.9* 63.2  30 μM Compound A pdp 8  7.3 ± 4.2*65.9 300 μM Compound A pdp 16   8.0 ± 3.0** 66.5 *p < 0.05, **p < 0.01

EXAMPLE 7

The procedures of Example 5 were repeated, but using various compoundsderived from Compound A by substitution at the α-amino group. Theresults are shown in the following Table 4.

TABLE 4 Compound n % Damage CA1 % Protection None (Hypoxia) 25 27.5 ±3.2  — 300 μM NαCBZ- 11  9.3 ± 4.3** 66.2 Compound A pre 300 μM NαCBZ-12  12.4 ± 4.3** 54.9 Compound A post None (Hypoxia) 57 21.3 ± 2.4  — 0.3 μM NαCBZ-  8 16.6 ± 7.2  22.1 Compound A post  3 μM NαCBZ- 15 16.7± 4.0  21.6 Compoumd A post  30 μM NαCBZ- 14 14.8 ± 4.5  30.5 Compound Apost 300 μM NαCBZ- 14  9.0 ± 2.7* 57.7 Compound A post None (Hypoxia) 3320.3 ± 2.4  — 300 μM Nαacetyl- 15 10.4 ± 4.0* 48   Compound A pre 300 μMNαacetyl- 20 16.3 ± 3.1  18.5 Compound A post None (Hypoxia) 49 26.8 ±4.1  — 300 μM Nαbenzyl-  8  6.4 ± 4.9** 76.1 Compound A post *p < 0.05,**p < 0.01 CBZ = benzyloxycarbonyl

EXAMPLE 8

The procedures of Example 5 were repeated, but using a compound(PyrAla3,4) in which the arginine of Compound A is replaced bypyridylalanine. The results are shown in the following Table 5.

TABLE 5 Compound n % Damage CA1 % Protection None (Hypoxia) 40 28.4 ±2.3 — 300 μM PyrAla3,4 post  7 29.9 ± 3.7 −5.3

It can be seen that this compound has a negative protective effect.

EXAMPLE 9

The procedures of Example 5 were repeated, but using either Compound I(in which the arginine of Compound A is replaced by citrulline) or acompound in which the arginine of Compound A is replaced by glutamine(Gln3,4). The results are shown in the following Table 6.

TABLE 6 Compound n % Damage CA1 % Protection None (Hypoxia) 16 23.2 ±4.9 — 300 μM Compound 1 post  7  10.9 ± 5.7* 53   300 μM Gln3,4 post  819.8 ± 6.5 14.7 *p < 0.05,

It can be seen that, whereas Compound I exerts a significant protectiveeffect, Gln3,4 has no such effect.

EXAMPLE 10

The procedures of Example 5 were repeated, but using either Compound Aor a compound corresponding to Compound A but in which R^(c) is atetramethylene group, i.e. the total number of carbon atoms in R^(b) andR^(c) is 8 (Arg4,4). The results are shown in the following Table 7.

TABLE 7 Compound n % Damage CA1 % Protection None (Hypoxia) 15 27.7 ±3.1 — 300 μM Compound A post  8  12.1 ± 3.5** 56.3 300 μM Arg4,4 post  726.7 ± 2.1  3.6 **p < 0.01

It can be seen that, whereas Compound A exerts a significant protectiveeffect, Arg4,4 has no such effect.

Conclusion of Examples

In summation, a number of spermidine (polyamine) based compounds weresynthesised using a novel solid phase approach and evaluated for theirprotective effects against hypoxia-induced neuronal damage inhippocampal slice cultures. The neuroprotective effects of 300 μML-arginylspermidine were dramatic with complete protection beingobserved when added pre-hypoxia. When added post hypoxia, protection wasobserved in a concentration-dependent manner with substantial protection(>70%) at 300 μM with an EC 50 of from 3–30 μM. L-lysinylspermidine andL-ornithylspermidine were also protective, although to a lesser extentthan the arginylspermidine. Significantly, the D-enantiomers of allthree compounds were substantially less active (if at all) in providingneuroprotective activity than the L-enantiomers.

The amalgamation of solid phase/combinatorial chemistry and in vitromodels of neuronal damage (e.g. ischaemia related damage) provide anexcellent means to synthesise and investigate large numbers ofpotentially neuroprotective compounds. This approach presents thepossibility of the generation of compounds which may profoundlyinfluence the treatment of severe neurological damage such as thatoccurring after stroke.

1. A method of treating a mammal to reduce incidences of neuronal celldeath caused by an ischaemic event by administering to said mammalbefore, after or during an ischaemic event an effective amount of asubstantially pure compound having the general formula (I)

wherein: Q represents an amidino group, a cyano group or a group offormula XYN—, where X and Y are the same or different, and each mayrepresent a hydrogen atom, a lower alkyl group, or hetero-atomcontaining group or, together with the nitrogen atom to which they areattached, form a nitrogen-containing heterocyclic group; R^(a)represents a straight or branched chain alkylene or alkenylene grouphaving from 1 to 6 carbon atoms and each optionally being substituted byfrom 1 to 4 alkyl groups each having from 1 to 3 carbon atoms; R^(b) andR^(c) each represent an alkylene or alkylene group having 3 or 4 carbonatoms in a straight chain, each being optionally substituted by a 1 or 2alkyl groups each having from 1 to 3 carbon atoms, the total number ofcarbon atoms in said straight chains of R^(b) and R^(c) being 7; R² andR³ are the same as or different from each other and each represents ahydrogen atom, or a group of formula R, RCO—, ROCO—, or RNHCO—, where Rrepresents a lower alkyl group or an aryl group, said alkyl or arylgroup being optionally substituted by one or more of the substituents α,defined below; the chiral carbon atom indicated by the asterisk is inthe L configuration; Z is an aromatic amino acid residue; n is 0 or 1;R¹ represents a hydrogen atom or a lower alkyl group or an aryl group,said alkyl or aryl group being optionally substituted by one or more ofthe substituents α, defined below; W represents a hydrogen atom or analkyl or aryl group; and substituents α are selected from: halogenatoms, amino groups, alkylamino groups, dialkylamino groups, cyanogroups, hydroxy groups, alkyl groups (except when the substituted groupis alkyl), aryl groups, carbamoyl groups, alkylcarbamoyl groups,dialkylcarbamoyl groups and carboxy groups and esters thereof; andpharmaceutically acceptable salts thereof.
 2. A method according toclaim 1, said compound having the formula (Ia):

wherein Q, R^(a), R^(b), R^(c), R², R³, Z, n, and R¹ are as in claim 1.3. A method according to claim 1, said compound having the formula (Ib):

wherein: X, Y, Z, n and R¹ are as defined in claim 1; x is an integerfrom 1 to 5; y is 3 or 4; R⁴, R⁵, R⁶ and R⁷ may be the same or differentand each represents a hydrogen atom or a lower alkyl group; and thechiral carbon atom indicated by the asterisk is in the L configuration.4. A method according to claim 1, in which Z represents an aromaticamino acid residue in the L configuration.
 5. A method according toclaim 1, wherein said compound is non-toxic.
 6. A method according toclaim 2, wherein said compound is non-toxic.
 7. A method according toclaim 3, wherein said compound is non-toxic.
 8. A method according toclaim 1, wherein said compound is:


9. A method according to claim 1, wherein said compound is:


10. A method according to claim 1, wherein said compound is:


11. A method according to claim 1, wherein said compound is:


12. A method according to claim 1, wherein said compound is:


13. A method according to claim 1, wherein said compound is:


14. A method according to claim 1, wherein said compound is:


15. A method according to claim 1, wherein said compound is:


16. A method according to claim 1, wherein said compound is:


17. A method of treating a mammal to reduce incidences of neuronal celldeath caused by an ischaemic event by administering to said mammal asubstantially pure compound having the general formula (I)

wherein: Q represents an amidino group, a cyano group or a group offormula XYN—, where X and Y are the same or different, and each mayrepresent a hydrogen atom, a lower alkyl group, or a simple hetero-atomcontaining group or, together with the nitrogen atom to which they areattached, form a nitrogen-containing heterocyclic group; R^(a)represents a straight or branched chain alkylene or alkenylene grouphaving from 1 to 6 carbon atoms and each optionally being substituted byfrom 1 to 4 alkyl groups each having from 1 to 3 carbon atoms; R^(b) andR^(c) represent an alkylene or alkenylene group having 3 or 4 carbonatoms in a straight chain, each being optionally substituted by 1 or 2alkyl groups each having from 1 to 3 carbon atoms, the total number ofcarbon atoms in said straight chains of R^(b) and R^(c) being 7; R² andR³ are the same as or different from each other and each represents ahydrogen atom, or a group of formula R, RCO—, ROCO—, or RNHCO—, where Rrepresents a lower alkyl group or an aryl group, said alkyl or arylgroup being optionally substituted by one or more of the substituents α,defined below; the chiral carbon atom indicated by the asterisk is inthe L configuration; Z is an aromatic amino acid residue; n is 0 or 1;R¹ represents a hydrogen atom or a lower alkyl group or an aryl group,said alkyl or aryl group being optionally substituted by one or more ofthe substituents α, defined below; W represents a hydrogen atom or analkyl or aryl group; and substituents α are selected from: halogenatoms, amino groups, alkylamino groups, dialkylamino groups, cyanogroups, hydroxy groups, alkyl groups (except when the substituted groupis alkyl), aryl groups, carbamoyl groups, alkylcarbamoyl groups,dialkylcarbamoyl groups and carboxy groups and esters thereof; andpharmaceutically acceptable salts thereof.
 18. The method according toclaim 17, said compound having the formula (Ia):

wherein Q, R^(a), R^(b), R^(c), R², R³, Z, n, and R¹ are as in claim 17.19. The method according to claim 17, said compound having the formula(Ib):

wherein: X, Y, Z, n and R¹ are as defined in claim 17; x is an integerfrom 1 to 5; y is 3 or 4; R⁴, R⁵, R⁶ and R⁷ may be the same or differentand each represents a hydrogen atom or a lower alkyl group; and thechiral carbon atom indicated by the asterisk is in the L configuration.20. The method according to claim 17, in which Z represents an aromaticamino acid residue in the L configuration.
 21. The method according toclaim 17, wherein said compound is non-toxic.
 22. The method accordingto claim 18, wherein said compound is non-toxic.
 23. The methodaccording to claim 19, wherein said compound is non-toxic.
 24. Themethod according to claim 17 wherein said compound is:


25. The method according to claim 17 wherein said compound is:


26. The method according to claim 17 wherein said compound is:


27. The method according to claim 17 wherein said compound is:


28. The method according to claim 17 wherein said compound is:


29. The method according to claim 17 wherein said compound is:


30. The method according to claim 17 wherein said compound is:


31. The method according to claim 17 wherein said compound is:


32. The method according to claim 17 wherein said compound is: